This invention relates to an extraction process for extracting carotenoids from carotenoid sources such as biomasses, which may be naturally-occurring or may be cultivated for the purpose. Such carotenoids find commercial application as pigments, anti-oxidants and food supplements, both for human and animal consumption.
Salmonids reared in the wild acquire a pink coloration resulting from the ingestion of micro-crustacea containing the red pigment astaxanthin as a natural part of their regular diet. When salmonids are reared in captivity, this is astaxanthin is artificially added to the feed in order to produce the desired pink coloration of the salmonid flesh. The amount of astaxanthin required to achieve this coloration is typically about 60 mg per kilogram of fish feed. Such pigments are also used to increase the skin colour of farmed fish such as sea bream and in shrimp farming.
Both synthetic and natural sources of astaxanthin have been used for salmonid pigmentation. The main source currently used is synthetic due to the high cost of natural-source formulations. Natural-source formulations, have included the direct incorporation of astaxanthin-rich biomass such as the yeast Pfaffia rhodozyma, the alga Haematococcus pluvalis, and crustacean products directly in the salmonid feed. However, said direct incorporation has not proved cost effective vis-à-vis the synthetic product due to the inability of the fish""s digestive system to effectively extract the astaxanthin from the biomass cells; i.e. the low bioavailability of this source.
Other commercially important carotenoids including lycopene and xcex2-carotene are also currently more expensive when extracted from natural sources than their synthetic counterparts.
Given the economic importance of astaxanthin and other carotenoids, numerous methods of optimizing the production of carotenoid-rich biomasses and improving carotenoid-extraction therefrom have been attempted.
The known natural sources of astaxanthin include shellfish, krill, the yeast P. rhodozyma, the green algae Haematococcus pluvialis and certain bacterial strains. Natural sources of lycopene include tomatoes and tomato waste, and carrots are a natural source of xcex2-carotene.
Due to the large volume of shrimp processing waste available this has seemed a promising source of astaxanthin. However the concentrations are low and the extraction methods attempted have proved expensive.
U.S. Pat. No. 3,906,112 describes the fine grinding of shrimp waste and the mixing and heating of the resulting powder with an oil in order to extract some of the astaxanthin into the oil.
U.S. Pat. No. 4,505,936 describes a similar process but where the chitinous shell is removed and the proteinaceous tissue is acidified before heating with oil.
More recent efforts have used supercritical CO2 to perform the extraction, and U.S. Pat. No. 5,210,186 describes the use of boiling lye to form an alkaline extract.
The common disadvantage to all these methods is that, given the low concentration of astaxanthin present and the high cost of extraction, the astaxanthin extracted is not cost-effective relative to the synthetic alternatives.
Culturing and extracting products via the fermentation of bacteria is well known in industry, and thus considerable effort has been extended in the direction of strain development to this end. U.S. Pat. No. 5,607,839 describes a bacterium belonging to a newly discovered genus that can produce carotenoids including astaxanthin. Similar strain development efforts have also been performed for microalgal sources. Nevertheless, both these sources are still far from commercialization due to the low yields obtained.
Much commercial effort has been expended in recent years on the strain development of the astaxanthin-producing yeast, P. rhodozyma. This work has succeeded in producing high yields of astaxanthin by superior strains of P. rhodozyma. For example, U.S. Pat. No. 5,599,711 describes a strain of this yeast capable of producing astaxanthin in the thousands of ppm range. However, despite the high yields achieved, this yeast has not proved cost-effective due to the following reasons:
1. When P. rhodozyma is added directly to the feed, the bioavailability is low due to its hard cell-wall which hampers the extraction of the astaxanthin molecule by the digestive system of the fish within the digestive time cycle. Attempts to mill or otherwise rupture the cell wall to enhance this bioavailability have only marginally improved the results.
2. The tight binding of the astaxanthin within the cell is the reason behind the relatively expensive nature of the methods for chemically extracting this material from the P. rhodozyma. For example, U.S. Pat. No. 5,356,810 discloses the extraction of astaxanthin from dried, unruptured P. rhodozyma cells using glacial acetic acid.
There is also extensive literature concerning the extraction of carotenoids in general.
U.S. Pat. No. 5,830,738 describes the use of enzymes to decompose cellular walls so as to extract carotenoids trapped in plant cells.
U.S. Pat. No. 5,789,647 described the use of compressed gases such as butane and propane together with organic entraining agents in order to extract carotenoids from natural materials.
U.S. Pat. No. 5,773,075 describes the use of high-temperature and pressure to extract carotenoids from plant material. However, all these methods are expensive and are thus unsuitable for extracting carotenoids from biomasses in a commercial application.
In accordance with the invention described below, a carotenoid is extracted from a carotenoid source using a mixture of water, a water immiscible solvent and a water soluble co-solvent.
U.S. Pat. No. 4,439,629 discloses extracting xcex2-carotene from algae by treating the algae at 50 to 100xc2x0 C. with calcium hydroxide to saponify the chlorophyll present and then extracting the xcex2-carotene with a solvent such as methylene chloride, hexane or high boiling petroleum ether.
U.S. Pat. No. 4,871,551 describes extracting astaxanthin from Haematococcus algae by extraction with solvents such as oils, aromatics (e.g. benzene), halogenated hydrocarbons (e.g. methylene chloride), or alkanes (e.g. hexane).
EP-A-612725 describes extraction of xcex2-carotene from halophilic algae of the genus Dunaliella including D. salina, D. parva, D. tertiolecta, D, primolecta and D. peircei. An aqueous suspension of the biomass is emulsified with edible oil (soya bean oil, peanut oil, or sunflower seed oil) at elevated temperatures and the mixture is subjected to membrane ultra-filtration.
WO 98/03480 describes the extraction of xcex2-carotene from algae (Dunaliella), vegetables (carrots), or fungi (Blakeslea) by extraction with a water immiscible solvent such as ethyl acetate, butyl acetate, hexane, or vegetable oil, followed by crystallisation and washing of the crystals with a poor solvent for xcex2-carotene, such as ethanol or ethyl acetate.
WO 98/50574 describes extraction of a carotene present as crystals in a biomass by disrupting the cells of the wet biomass, possibly by using a solvent such as octanol, optionally adding a water immiscible solvent which may be oil, hexane or ethyl acetate, and removing debris, to leave solid carotene floating over liquid. The solid upper layer may be washed with water and then with a poor solvent for the carotenoid, such as methanol, ethanol, isopropanol or acetone to remove lipid.
Extraction of xcex2-carotene from natural sources using as solvent one of acetone, methyl-ethyl ketone, methanol, ethanol, propan-2-ol, hexane, dichloromethane and super-critical carbon dioxide is described in U.S. Pat. No. 5,714,658, as is the use of a mixture of an acetic acid ester, such as ethyl acetate, and an edible oil. This however is a combination of two water immiscible solvents.
An extraction protocol for use in an assay for the content of astaxanthin in fish feed is described in xe2x80x9cAnalytical Methods for Vitamins and Carotenoids in Feedxe2x80x9d Ed: P. Hofmann, H. E. Keller, J. Schierle and W. Schxc3xcep, Dept. of Vitamin Research and Developmentxe2x80x94Roche Basel. This involves extraction with a single phase medium of dichloromethane and ethanol.
In summary, although a high-yield natural source for astaxanthin (optimal strains of P. rhodozyma) has been developed, it is not currently cost-effective as a salmonid feed additive as the prior art in extraction has not enabled this astaxanthin to be inexpensively extracted and formulated. Similarly, efforts to extract other carotenoids from natural sources have not yet been implemented in a cost-effective manner relative to the cost of their synthetic analogs.
The present invention now provides a process for the extraction of a carotenoid from a carotenoid source comprising treating the carotenoid source at an elevated temperature with a multi-phase solvent mixture comprising water, a hydrophobic carotenoid solvent and a water soluble co-solvent so as to extract the carotenoid from the carotenoid source into the hydrophobic solvent.
Water may be added or may be present naturally in the carotenoid source. The multiphase mixture will of course generally consist of two phases, one mainly aqueous and the other mainly non-aqueous or oily.
The process is preferably carried out at a temperature of from 50xc2x0 C. upwards, for instance at a temperature of from 50 to 80xc2x0 C., more preferably from 60 to 70xc2x0 C.
The biomass is preferably agitated, e.g. stirred, during the treatment.
The extraction is carried on for a suitable period, but preferably for at least 30 minutes, for instance for from 30 minutes to 3 hours, preferably for about 1 hour.
The hydrophobic solvent is preferably chosen to combine its hydrophobic properties with good solvent abilities for the carotenoid and is suitably an edible oil, methyl acetate, ethyl acetate, butyl acetate, a C5 or above alkane or chloroform.
The water soluble co-solvent is preferably chosen to be water soluble and to have at least some limited ability to dissolve the carotenoid, but such that the partition coefficient of the carotenoid between the solvent and the co-solvent is such that carotenoid dissolved in the co-solvent is transferred to the solvent. Suitably the co-solvent is an alcohol, a ketone, an ether, or a cyclic ether. Preferably, the water soluble co-solvent is methanol, ethanol, n-propanol or iso-propanol, or is a monoalkyl ester of ethylene glycol (cellosolve), or is 1,3-dioxane or 1,4-dioxane.
A solution of carotenoid in edible oil may be obtained directly by using the edible oil as the solvent but in some circumstances it is advantageous that the hydrophobic solvent is not an edible oil and that following said extraction, the hydrophobic solvent is mixed with edible oil and is evaporated therefrom. The evaporated hydrophobic solvent is preferably then re-used in a said extraction process.
The carotenoid may be astaxanthin, lycopene or beta-carotene, by way of example.
A major field of applicability of the invention is where the carotenoid source is a biomass, such as a yeast of the genus Phaffia, shell fish, shell fish waste, krill, algae, fungi, vegetable, or tomato.
However, the process may be used generally for bringing a carotenoid into solution, especially solution in edible oil, and so said carotenoid source may for instance be a carotenoid synthesis reaction mixture or a solid carotenoid obtained by synthesis or previous extraction from a natural source. The invention therefore includes a process of carotenoid synthesis producing a reaction mixture from which a carotenoid is extracted in a work-up procedure including an extraction process as described herein.
The invention further includes a process for preparing a solution of a carotenoid in an edible oil, comprising mixing a solution of said carotenoid in a volatile, water immiscible solvent with said edible oil and evaporating the water immiscible solvent from said mixture.
The invention further provides a process for producing a feed material comprising carrying out a carotenoid extraction process as previously described, so as to provide a solution of carotenoid in edible oil, and adding said solution to feed ingredients.
Preferred features of the invention will be described below. The following discussion and exemplification mostly refers to the extraction and formulation of astaxanthin, although the general application of this method to other carotenoids will be readily understood by one skilled in the art.
Suitably, an aqueous suspension of biomass is heated together with water and an ethanol co-solvent in contact with a hydrophobic solvent such as an consumable oil in which astaxanthin or other carotenoids is readily dissolved. Without wishing to be limited by theory, the water-ethanol mixture serves to: (a) disassociate the protein-carotenoid bonds holding the carotenoid within the cell and (b) remove the carotenoid through the cell wall of the biomass. As astaxanthin is lipophilic and thus is more soluble in a hydrophobic solvent such as oil than in the aqueous co-solvent, the astaxanthin rapidly moves from the aqueous co-solvent to the oil, where this astaxanthin is accumulated. To optimize the contact between the aqueous co-solvent and the oil, the solution is vigorously stirred, thus creating an emulsion. By then separating out the oil layer from the aqueous suspension layer, the astaxanthin is removed in an oil formulation.
The hydrophobic solvent for the astaxanthin may be either an oil or a volatile compound such as ethyl acetate. If it is desired to increase the concentration of astaxanthin in the eventual oil formulation, a two-step process may be used as follows: (1) Ethyl acetate serves as the hydrophobic solvent in the astaxanthin extraction as above, (2) After separation of the layers the astaxanthin-bearing ethyl acetate is mixed in a further vessel with oil and the ethyl acetate is then evaporated off, transferring the astaxanthin to the oil. By recycling such an alternative hydrophobic solvent several times through the oil, the astaxanthin concentration achieved in said oil may thus be increased significantly, thereby advantageously reducing transportation costs.
The astaxanthin-rich oil produced by the process of the present invention may be directly used as an additive to salmonid fish feed, preferably after the addition of suitable anti-oxidants. Bioavailability testing of said additive showed that, advantageously, this astaxanthin-rich oil has a higher bioavailability than that of a commercially-available synthetic product (xe2x80x9cCarophyl Pinkxe2x80x9d, from Hoffman La-Roche) and a much higher bioavailability than that of dried P. rhodozyma cells.
The carotene is preferably a xanthophyll carotene, i.e. a carotene containing oxygen containing groups (xe2x80x94OH, xe2x80x94Oxe2x80x94, or xe2x95x90O).
This process, and its principle of operation will be better understood by reference to the following specific examples: